1Helen Diller Family Comprehensive Cancer Center and Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.

Abstract

Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia (AML). It has been hypothesized that gain of the MYC protooncogene is of central importance in trisomy 8, but the experimental data to support this are limited and controversial. In a mouse model of promyelocytic leukemia in which the MRP8 promoter drives expression of the PML-RARA fusion gene in myeloid cells, a Myc allele is gained in approximately two-thirds of cases as a result of trisomy for mouse chromosome 15. We used this model to test the idea that MYC underlies acquisition of trisomy in AML. We used a retroviral vector to drive expression of wild-type, hypermorphic, or hypomorphic MYC in bone marrow that expressed the PML-RARA transgene. MYC retroviruses cooperated in myeloid leukemogenesis and suppressed gain of chromosome 15. When the PML-RARA transgene was expressed in a Myc haploinsufficient background, we observed selection for increased copies of the wild-type Myc allele concomitant with leukemic transformation. In addition, we found that human myeloid leukemias with trisomy 8 have increased MYC. These data show that gain of MYC can contribute to the pathogenic effect of the most common trisomy of human AML.

MYC mutants cooperate with PML-RARα to induce AML. (A) Bone marrow of PML-RARA (PR) or control FVB/n (Cntr) mice was transduced with a retrovirus encoding MYCT58A and introduced into lethally irradiated recipient mice. Combined results from two independent experiments for each group are shown. Median time to APL of 10 PML-RARα + MYCT58A mice was 70 d. Control FVB/n + MYCT58A mice developed AML (5 mice), T-ALL (3 mice), or were euthanized without evidence of leukemia or lymphoma (2 mice). Median time to disease was 101 d. Difference in leukemia-free survival: P < 0.0001. (B) Bone marrow of PML-RARA (PR) or control FVB/n (Cntr) mice was transduced with a retrovirus encoding MYC with a deletion of the MBII domain (MYCΔMBII) and introduced into lethally irradiated recipient mice. Combined results from two independent experiments for each group are shown. Median time to APL of 10 PML-RARα + MYCΔMBII mice was 92 d. Control FVB/n + MYCΔMBII mice developed T-ALL (four mice) or were euthanized without evidence of leukemia or lymphoma (six mice). Difference in leukemia-free survival: P < 0.0001.

Southern blot of PML-RARα + MYC leukemias shows clonal retroviral integrations. Genomic DNA samples were digested with EcoRI, which cuts within the multicloning site of retroviral integrants, and the blot was probed with a probe hybridizing to GFP sequences. 6989, A GFP+ leukemia that arose in a PML-RARα + MIG recipient mouse. 34, A GFP+ lymphoblastic lymphoma that arose in a recipient of Control + MYC-transduced bone marrow. PR+MYC Pre, preleukemic bone marrow from PML-RARα + MYC mice 5 wk after transplantation. PR+MYC, PR+MYCΔMBII, and PR+MYCT58A, leukemias that arose from recipients of PML-RARα bone marrow transduced with various MYC alleles. Data in this figure were obtained in three independent Southern blots. Thick vertical lines separate groups of samples and indicate juxtapositions of lanes. Thin vertical lines also indicate juxtaposition of lanes.

Haploinsufficiency for Myc delays leukemia development. (A) Bone marrows of PML-RARA Myc+/+ or PML-RARA Myc+/− FVB/n mice were harvested and transplanted into lethally irradiated FVB/n recipient animals. Combined results from eight (Myc+/+) or eleven (Myc+/−) independent transplantation experiments are shown. Mice were followed for the development of leukemia. Nonleukemic deaths were censored at the time of death. 67% of PML-RARA Myc+/+ deaths were from leukemia, whereas 31% of PML-RARA Myc+/− recipient mice died of leukemia. Median latency among leukemic animals was 258 d for Myc+/+ and 339 d for Myc+/−. P < 0.0001. (B) Morphology of leukemias arising in PML-RARA Myc+/− mice. Results representative of 16 leukemias are shown. (i) Cytology of leukemic cells from spleen of mouse #836. L, Lymphocyte. Histology of (ii) bone marrow of mouse #828, (iii) spleen of mouse #828, and (iv) liver of mouse #828. (i) Wright’s Giemsa stain. (ii-iv) H&E stain. Bars: (i) 8 µm; (ii) 12 µm; (iii and iv) 60 µm; (iii inset) 24 µm. (C) Gain of chromosome 15 and the wild-type Myc allele in PML-RARA Myc+/− leukemias. The number of copies of chromosome 15 as determined by cytogenetic analysis is indicated for each sample; 2 previously characterized leukemias (#1111 and #1127), 5 PR+Myc+/− leukemias, and 1 nonleukemic PR+Myc+/− marrow. Copy numbers for the wild-type Myc and Pgk-hprt alleles are also given for the same samples. Samples were run in triplicate in one to eight independent experiments. Pgk-hprt copy number could not be determined for leukemia #5727 because of insufficient quantity of DNA. *, Pgk-hprt copy number values are 0. Results for leukemia #836 showing no gain of chromosome 15, but increased copy-number for the wild-type Myc allele, are not shown here, but are included in Table I and discussed in the text.